The objective of the proposed research is to establish in vitro conditions for the assembly of functionally active E. coli 30S ribosomes from precursor 16S RNA (p16S RNA). In particular, we have been investigating the role of ribosomal proteins in the methylation of p16S RNA. Methylation of p16S RNA requires addition of a partially purified "enzyme" fraction from E. coli. Some methylation occurs in the absence of ribosomal proteins, but is stimulated approximately 3-fold by addition of 30S ribosomal proteins. The 30S ribosomal proteins which stimulate most strongly (S5, 7, 8, 9, 10, 12, 13, 14, and 19) are proteins which have previously been shown to bind near the middle and 3' end of 16S RNA. These results indicate that the ribosomal proteins which stimulate methylation of p16S RNA bind near or at the site of methylation and that methylation occurs during an intermediate stage(s) of ribosome assembly.